Summary T lymphocytes use the T cell receptor (TCR) to recognize antigens in the form of peptide-major histocompatibility complexes (pMHCs) on antigen-presenting cells (APCs). CD8 is a co-receptor that binds unliganded MHC with low affinity but TCR-liganded pMHC with 2-logs higher affinity. Some pMHCs behave as null pMHCs by themselves but as coagonist pMHCs when co-presented with agonist pMHCs by greatly enhancing TCR responses. While both phenomena have been reported, their mechanisms remain elusive due to the lack of tools to analyze multimeric interactions among multiple receptor-ligand species quantitatively. We propose to design and construct multi-DNA force probes to quantitatively study force generation and sensing by T cells. In particular, we will study how T cells exert differential forces via the TCR and CD8 on dual species of pMHCs that are clustered on the APC surface to promote multivalent binding. Using our expertise in DNA nanotechnology, we will create various DNA origami based tension probes that will report the TCR- and CD8-pMHC binding events in a quantitative fashion. The proposed experiments will be carried out in the following specific aims. (1) To develop multi-color DNA force probes for studying the cooperative effect of coagonist pMHC on agonist pMHC binding to TCR and CD8. Dual color DNA-force probes will be designed to report the binding of agonist and coagonist independently. Two sets of experiments will be performed using wild-type MHC that allows CD8 binding and/or mutant MHC that prevents CD8 binding to present agonist and/or coagonist peptides in different combinations. Our study will provide insights to how coagonist binding to CD8 affects agonist binding in terms of force generation and sensing by the T cell. (2) To develop DNA force probe arrays with controlled coagonist/agonist spatial organization for investigating the multivalent binding between TCR/CD8 and agonist/coagonist. We will create complex arrays of dual color probes to simulate the clustering effect. Origami- based DNA nanostructures with various inter-probe distances, ratios and densities will be used to investigate the clustering effect. Our study will shed light on the mechanism of T cell mechanosensing. T cell functions are governed by coordinated interactions of many receptors, including antigen receptor/coreceptor, adhesion receptors, cytokine receptors, and co-stimulatory/inhibitory receptors. The tools developed herein are applicable to study of other molecular interactions of T cells and other cells in the immune system or other biological systems, thereby potentially impacting the broader biomedicine field where cell surface molecular interactions are being targeted for treatment of various diseases, including autoimmunity, viral infection, and cancer. !